Molecular Mechanisms Responsible for Therapeutic Potential of Mesenchymal Stem Cell-Derived Secretome

Mesenchymal stem cell (MSC)-sourced secretome, defined as the set of MSC-derived bioactive factors (soluble proteins, nucleic acids, lipids and extracellular vesicles), showed therapeutic effects similar to those observed after transplantation of MSCs. MSC-derived secretome may bypass many side effects of MSC-based therapy, including unwanted differentiation of engrafted MSCs. In contrast to MSCs which had to be expanded in culture to reach optimal cell number for transplantation, MSC-sourced secretome is immediately available for treatment of acute conditions, including fulminant hepatitis, cerebral ischemia and myocardial infarction. Additionally, MSC-derived secretome could be massively produced from commercially available cell lines avoiding invasive cell collection procedure. In this review article we emphasized molecular and cellular mechanisms that were responsible for beneficial effects of MSC-derived secretomes in the treatment of degenerative and inflammatory diseases of hepatobiliary, respiratory, musculoskeletal, gastrointestinal, cardiovascular and nervous system. Results obtained in a large number of studies suggested that administration of MSC-derived secretomes represents a new, cell-free therapeutic approach for attenuation of inflammatory and degenerative diseases. Therapeutic effects of MSC-sourced secretomes relied on their capacity to deliver genetic material, growth and immunomodulatory factors to the target cells enabling activation of anti-apoptotic and pro-survival pathways that resulted in tissue repair and regeneration.


Introduction
Many degenerative and inflammatory diseases are in the focus of stem cell-based research. Among different populations of stem cells, mesenchymal stem cells (MSCs) represent the most promising resource for the cell-based therapy of inflammatory and degenerative diseases on the ground of their multi-lineage differentiation potential, immuno-modulatory properties and pro-angiogenic characteristics [1][2][3][4][5][6]. MSCs spontaneously differentiate into osteoblasts, chondrocytes and adipocytes regulating normal turnover and homeostasis of adult mesenchymal tissues [7,8]. Importantly, MSCs have a differentiation potential broader than initially thought. Under strictly defined in vitro conditions, which results in up-regulation of Fas-associated phosphatase-1 (Fap-1) and caveolin-1 (Cav-1) in MSCs. Fas binds to Fap-1 and Cav-1 and activates Soluble N-ethylmaleimide-sensitive factor (NSF) Attachment protein Receptor (SNARE)-mediated membrane fusion resulting in enhanced secretion of IL-1Ra-bearing Exos in extracellular space. In this manner, TNF-alpha-primed MSCs, through the delivery of IL-1Ra-containing MSC-Exos, inhibit IL-1β:IL-1R signaling and protect tissues from inflammation-induced injuries [56].
In line with these findings, it was recently revealed that MSCs exposed to inflammatory cytokines (TNF-α and IFN-γ) generate MSC-CM and MSC-Exos with enhanced immunomodulatory properties [59]. IFN-γ and TNF-α provoke MSCs to express inducible nitric oxide synthase (iNOS) which increases IDO-1 activity in MSCs. Accordingly, administration of MSC-CM, in iNOS and IDO-1/KYN-dependent manner suppressed inflammatory and cytotoxic potential of T lymphocytes and NKT cells [5].
In line with these findings, PB-MNCs which were cultured in the presence of TNF-α and IFN-γ-stimulated MSC-Exos, produced lower amounts of 34 inflammation-related cytokines and chemokines, but significantly increased secretion of several anti-inflammatory mediators, including IL-10 [59]. IL-10 inhibits maturation, down-regulates expression of co-stimulatory molecules and attenuates antigen-presenting function of DCs which results in suppression of T cell-driven inflammation [60]. TNF-α and IFN-γ-priming significantly increased concentration of PGE2 in MSC-CM and MSCs-Exos [59]. PGE2 has an important role in immunosuppression mediated by MSC-derived secretomes [43,60]. PGE2 has direct inhibitory effects on IL-2 production and attenuates expression of janus kinase (Jak)-3 which mediates the responsiveness of T cells to IL-2 [61]. Accordingly, MSC-CM and MSCs-Exos, in a PGE2-dependent manner, suppressed clonal expansion of activated T cells and attenuated T cell-driven inflammation [5,60,62]. Additionally, through the secretion of PGE2, MSC-sourced CM and Exos favored alternative activation of macrophages, prevented maturation of DCs and suppressed cytotoxicity of NK and NKT cells [5]. NK and NKT cells, cultured in the presence of PGE2-containing MSC-CM, failed to optimally express cytotoxic molecules and significantly reduced production of inflammatory cytokines (TNF-α, IFN-γ and IL-17) upon activation [5,43].
An enhanced immunosuppressive property of TNF-α and IFN-γ-primed MSC-Exos are in line with previously published data showing that MSCs have a dynamic response to local microenvironment [5]. As far as we know to date, MSCs are not constitutively immunosuppressive. They alter their secretory profile and immunomodulatory characteristics in dependence of inflammatory milieu to which they are exposed. In the presence of low concentration of IFN-γ and TNF-α, MSCs obtain pro-inflammatory phenotype and produce large amounts of inflammatory cytokines and chemokines that stimulate activation and migration of immune cells in inflamed tissues. On the contrary, when MSCs are exposed to the high levels of inflammatory cytokines, they adopt anti-inflammatory phenotype and secrete immunosuppressive factors that inhibit generation of inflammatory M1 macrophages, maturation and antigen-presenting function of DCs, effector functions of inflammatory CD4+Th1, CD4+Th17 cells, CD8+ cytotoxic T lymphocytes (CTLs), NK and NKT cells [5]. In line with these findings, IFN-γ and TNF-α-priming of MSCs should be used to promote generation of MSC-Exos with enhanced immunosuppressive properties that could have better therapeutic effects in the treatment of autoimmune and inflammatory diseases.

The Role of MSC-Sourced Secretome in Tissue Repair and Regeneration
In addition to immunosuppressive cytokines, MSC-sourced secretome also contains cocktail of growth factors which promote tissue repair and regeneration, wound healing and neo-angiogenesis. Elevated concentration of tissue inhibitors of metalloproteinase (TIMP)-1 and 2, fibroblast growth factor (FGF)-6 and 7 and HGF were considered responsible for beneficial effects of MSC-CM in corneal epithelial wound healing [63]. Similarly, HGF-containing MSC-CM was involved in liver repair, regeneration [64], MSC-derived neurotrophins (brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF)), which were crucially important for MSC-CM-based alleviation of spinal cord injury [65].
MSC-CM-induced anti-fibrotic and angiomodulatory effects were also responsible for enhanced wound healing and reduced scar formation in MSC-CM treated animals [37]. Concentration of pro-and anti-angiogenic factors in MSC-CM is regulated by inflammatory and hypoxic conditions to which MSCs were exposed. When MSCs are cultured in the presence of high concentration of inflammatory cytokines and engraft in the inflammatory microenvironment, they start to produce anti-angiogenic molecules in order to prevent migration of circulating leucocytes in the inflamed tissues [37]. An extensive proteomic analysis of MSC-CM revealed that MSC-derived TIMP-1 was mainly responsible for anti-angiogenic effects of MSC-sourced secretome [66].
Several recently published studies revealed that MSC-derived secretome was also able to regulate apoptosis in physiological and pathological conditions. MSC-CM-based therapy significantly decreased expression of pro-apoptotic Bax and cleaved caspase-3 but increased expression of anti-apoptotic Bcl-2 in parenchymal cells, preventing their loss during on-going inflammation [37]. Interestingly, completely opposite effects were noticed in MSC-CM-treated tumor cells. Significantly increased activity of caspase-3, -8, -9, -12 were noticed in MSC-CM-treated MDA-MB-231 breast cancer cells [71]. Importantly, these findings were also confirmed in vivo, in a xenograft mouse tumor model. MSC-CM treatment resulted in significantly reduced breast cancer growth and increased survival of tumor-bearing mice [71]. However, it should be noted that these beneficial effects were only observed in tumor bearing mice that received human uterine cervical MSC-derived conditioned medium (hUTC-MSC-CM) while treatment with human adipose tissue MSC-derived conditioned medium (hAT-MSC-CM) did not result in attenuated tumor growth. Compared to hAT-MSC-CM, hUCT-MSC-CM contains high levels of factors which induce apoptosis of tumor cells (tumor necrosis factor superfamily member 14 (TNFSF14) and promote anti-tumor Th1 cell-mediated immune response (C-X-C motif chemokine ligand (CXCL)10 and Fms-related tyrosine kinase 3 ligand) [37]. On the other hand, umbilical cord (UCD)-MSC-CM contains low levels of factors which promote tumor growth (epidermal growth factor receptor (EGFR), FGF-4 and -9), neo-angiogenesis (VEGF, IL-6, IL6 receptor), homing of naïve T cells to peripheral lymph nodes (chemokine (C-C motif) ligand 7 (CCL7)) and migration of circulating monocytes in tumor tissue (macrophage migration inhibitory factor (MMIF)) [37]. These findings indicate that MSC-CM-based effects depend on MSCs origin and suggest that content of MSC-CM should be precisely determined before clinical application.

Experimental Evidence for Therapeutic Potential of MSC-Derived Secretome in the Treatment of Inflammatory and Degenerative Diseases
A large number of experimental studies explored therapeutic potential of MSC-sourced secretome and their findings indicated that MSC-CM and MSC-Exos managed to efficiently enhance endogenous healing process in inflamed tissues by providing pro-angiogenic and trophic factors to injured cells, and by suppressing detrimental local and systemic immune response . MSC-derived secretome showed beneficial effects in the treatment of inflammatory and degenerative diseases of hepatobiliary, respiratory, skeletal, gastrointestinal, cardiovascular and nervous system .
By using several animal models of ALF, we and others demonstrated that administration of MSC-CM significantly improved liver regeneration and increased survival rate of experimental animals by reducing influx of inflammatory cells in the inflamed liver and by attenuating apoptosis and increasing proliferation of injured hepatocytes [15][16][17][72][73][74][75][76]. Importantly, therapeutic effects of MSC-CM were similar to those observed after transplantation of their parental MSCs [74][75][76]. Monitoring of adoptively transferred leukocytes revealed that a reduced influx of circulating immune cells in injured livers was a consequence of MSC-CM-mediated down-regulation of chemokine receptors (CXCR3 and CCR5), responsible for the trafficking of IFN-γ and IL-17-producing inflammatory T cells [15,75]. Among various numbers of MSC-derived immunomodulatory, trophic and hepatoprotective factors, MSC-CM-induced prevention of apoptosis and enhanced regeneration of injured hepatocytes was mainly mediated by IDO-1/KYN, HGF, fibrinogen-like protein 1 and IL-6/gp130 signaling pathways [15,16,74,76].
In line with these findings, we recently demonstrated that bone marrow (BM)-MSC-CM, in an IDO-1/KYN-dependent manner, efficiently alleviated ALF in mice by suppressing pro-inflammatory and cytotoxic potential of liver NKT cells, the main effector cells in fulminant hepatitis [74,76]. Attenuated expression of apoptosis-inducing ligands was observed on MSC-CM-treated liver NKT cells and was accompanied with reduced cytotoxicity of NKT cells against hepatocytes in vitro and in vivo. Additionally, MSC-CM-treated liver NKT cells had reduced capacity for production of inflammatory cytokines (TNF-α, IFN-γ, IL-4) and secreted higher amount of immunosuppressive IL-10 [76]. An addition of 1-methyl-dl-tryptophan (1-MT), a specific IDO-1 inhibitor, or l-N G -monomethyl arginine citrate, a specific inhibitor of inducible nitric oxide synthase (iNOS), completely abrogated immunosuppressive and hepatoprotective effects of MSC-CM and restored hepatotoxicity of NKT cells, suggesting that MSC-CM-mediated suppression of NKT cells was iNOS and IDO-1-dependent [76]. Having in mind that MSC-derived IDO-1 prevents trans-differentiation of Tregs in Th17 cells, we analyzed capacity of MSC-CM to regulate ratio between FoxP3-expressing, IL-10 producing immunosuppressive NKTreg cells and RORγT-expressing, IL-17-producing inflammatory NKT17 cells in the liver. Systemic administration of MSC-CM significantly reduced the total number of liver-infiltrating NKT17 cells and promoted expansion of liver NKTregs which resulted in attenuation of ALF [74]. Therapeutic potential of MSC-CM was completely abrogated by 1-MT, confirming the crucial importance of IDO-1/KYN pathway for immunosuppressive and hepatoprotective effects of MSC-CM in modulation of ALF [76].
In addition to IL-10, NGF also had important role in MSC-CM-induced suppression of HSCs [79]. Activated HSCs express p75 receptor, which, upon NGF stimulation, triggers apoptosis in these cells. Accordingly, MSC-CM in NGF/p75 dependent manner induced apoptosis of HSCs and alleviated liver fibrosis [79].
Several research groups indicated that MSC-Exos attenuated liver fibrosis by suppressing collagen production in HSCs [13,80,82]. Li and colleagues demonstrated that human UCD-MSC-Exos inhibited phosphorylation of Smad2 in HSCs, suppressed TGF-β/Smad2 signaling and attenuated synthesis of collagen type 1 and 3 [80]. Hyun and coworkers highlighted the importance of microRNA (miR)-125b-bearing MSC-Exos which suppressed pro-fibrotic function of HSCs by impeding activation of Hedgehog/Smoothened signaling pathway in these cells [82]. In line with these findings, Lou and associates suggested that miR-122 had a crucially important role in suppression of HSC-mediated liver fibrosis. By delivering miR-122 into HSCs, AT-MSC-Exos down-regulated expression of P4HA1 and IGF1R genes which controlled collagen production in HSCs [13].
Several research groups [84][85][86] demonstrated that administration of MSC-CM and MSC-Exos alleviated inflammation and airway remodeling in asthmatic animals ( Figure 1). De Castro and co-workers demonstrated that human AT-MSCs-Exos significantly attenuated ovalbumin (OVA)-induced allergic asthma in immunocompetent mice. AT-MSCs-Exos down-regulated total number of lung-infiltrated eosinophils and reduced expression of pro-fibrotic TGF-β in asthmatic lungs which resulted in decreased collagen fiber deposition [85]. Cruz and colleagues showed that systemic injection of BM-MSC-CM or BM-MSC-Exos significantly attenuated influx or inflammatory neutrophils, eosinophils, lymphocytes and macrophages in asthmatic murine lungs [84]. Importantly, BM-MSC-CM and BM-MSC-Exos were more potent than BM-MSCs in reducing the total number of neutrophils and eosinophils in the lungs. Additionally, BM-MSC-sourced secretome altered phenotype and function of lung-infiltrated antigen-specific CD4+T cells, resulting in attenuated airway inflammation. Detrimental Th2 and Th17 cell-driven inflammatory response in asthmatic lungs were suppressed by BM-MSC-derived secretome as evidence by significantly reduced number of IL-4, IL-5, and IL-17-producing CD4+T cells. Additionally, systemic administration of BM-MSC-sourced secretome increased total number of lung-infiltrated IL-10-producing CD4+ T cells and created immunosuppressive microenvironment in the lungs that allowed for better functional recovery of asthmatic animals [84]. Similar conclusions were made by Du and colleagues who confirmed in clinical settings that MSCs-Exos were able to successfully alleviate airway inflammation in asthmatic patients by modulating expansion and effector function of CD4+T cells [86]. MSC-Exos significantly attenuate antigen-presenting function of DCs and reduced their capacity for activation of naïve CD4+ T cells. Additionally, MSC-Exos promoted production of anti-inflammatory IL-10 and TGF-β in PB-MNCs of asthmatic patients and enhanced proliferative and immunosuppressive properties of Tregs [86].
Several lines of evidence suggested that beneficial effects of MSC-CM in COPD were a consequence of MSC-CM-induced inhibition of alveolar cell apoptosis or MSC-CM-based suppression of T cell:macrophage crosstalk in the lungs [23]. Administration of BM-MSC-CM reversed cigarette smoke-induced changes in caspase-3, p53, p21, p27, Akt, and p-Akt expression, suppressed collagen deposition and restored repair function of fibroblasts in the rat lungs [24]. MSC-CM-induced repair of injured alveolar epithelial cells was mainly relied on regenerative capacity of MSC-derived FGF-2 [23]. In line with these findings, Kim and colleagues designed FGF-2-bearing AT-MSC-sourced artificial nanovesicles which managed to efficiently alleviate COPD in mice by inducing proliferation of alveolar epithelial cells [25]. Importantly, lower doses of AT-MSC-derived artificial nanovesicles had beneficial effects similar to higher doses of AT-MSC-derived natural Exos, indicating that FGF-2-bearing artificial nanovesicles should be further explored in MSC-based cell-free therapy of COPD [25].
10 airway inflammation. Detrimental Th2 and Th17 cell-driven inflammatory response in asthmatic lungs were suppressed by BM-MSC-derived secretome as evidence by significantly reduced number of IL-4, IL-5, and IL-17-producing CD4+T cells. Additionally, systemic administration of BM-MSCsourced secretome increased total number of lung-infiltrated IL-10-producing CD4+ T cells and created immunosuppressive microenvironment in the lungs that allowed for better functional recovery of asthmatic animals [84]. Similar conclusions were made by Du and colleagues who confirmed in clinical settings that MSCs-Exos were able to successfully alleviate airway inflammation in asthmatic patients by modulating expansion and effector function of CD4+T cells [86]. MSC-Exos significantly attenuate antigen-presenting function of DCs and reduced their capacity for activation of naïve CD4+ T cells. Additionally, MSC-Exos promoted production of anti-inflammatory IL-10 and TGF-β in PB-MNCs of asthmatic patients and enhanced proliferative and immunosuppressive properties of Tregs [86]. Administration of MSC-sourced secretome significantly reduced influx of circulating eosinophils, neutrophils, monocytes and lymphocytes in asthmatic lungs resulting in alleviation of on-going inflammation. MSC-CM or MSC-Exos reduced TGF-β production, decreased collagen deposition and attenuated fibrosis in the lungs. Additionally, MSC-derived secretome attenuated antigen-presenting function of DCs and suppressed Th2 and Th17 cell-driven inflammatory response in asthmatic lungs, but increased total number of lung-infiltrated IL-10-producing Tregs which created immunosuppressive microenvironment that allowed better functional recovery of asthmatic animals.
Although MSCs can be used for the attenuation of chronic lung inflammation and fibrosis, plenty of evidence suggest that aberrant activation of Wnt/β-catenin and TGF-β signaling pathways in lung resident MSCs may induce their differentiation in miofibroblasts and could, consequently, contribute to the development of IPF [87]. Having in mind that beneficial effects of MSCs in the therapy of IPF were mainly relied on their paracrine effects [88], Shantu and colleagues investigated whether MSC-sourced secretome may attenuate IPF as efficiently as MSCs [89]. They demonstrated that human BM-MSC-derived EVs managed to attenuate IPF by suppressing TGF-β-induced myofibroblastic differentiation of lung fibroblasts [89]. Thy-1-integrin interaction-dependent pathway was crucially important for the delivery of MSC-EVs components in the fibroblasts. Human BM-MSC-EVs are enriched with miRNAs with anti-fibrotic and immunomodulatory properties, including miR-199a/b-3p, 21-5p, 630, 22-3p, 196a-5p, 199b-5p, 34a-5p and 148a-3p [90]. Among them, miR-630, was mainly responsible for suppression of pro-fibrotic genes in lung fibroblasts. Administration of miR-630containing MSC-EVs significantly reduced α-smooth muscle actin expression in lung fibroblasts and contributed to the MSC-EV-mediated alleviation of IPF [89,91].

Therapeutic Potential of MSC-Derived Secretome in Cartilage Regeneration
Several lines of evidence suggested that beneficial effects of MSC-based therapy of osteoarthritis (OA) are, at least partially, mediated by MSC-sourced secretome (Table 3) [26,27,[92][93][94][95][96]. Chen and colleagues showed that MSC-CM-treated chondrocytes significantly reduced production of inflammatory cytokines (TNF-α, IL-1β, IL-6), which play detrimental role in cartilage degeneration during OA development and progression ( Figure 2) [92]. These findings are in line with results reported by Tofino-Vian and co-workers who demonstrated that IL-1β-activated OA chondrocytes were not capable to optimally produce inflammatory mediators (TNF-α, IL-1β, IL-6 and nitric oxide (NO)) in the presence of human AT-MSC-CM or AT-MSC-Exos [93]. Even more, AT-MSC-CM and AT-MSC-Exo-treatment enhanced production of immunosuppressive IL-10 in IL-1β-activated OA chondrocytes, indicating anti-inflammatory and chondroprotective effects of AT-MSC-derived secretome [93]. Chondrocytes cultured in the presence of MSC-derived secretome significantly reduce production of inflammatory cytokines which play detrimental role in cartilage degeneration during OA development (TNF-α, IL-1β, IL-6, nitric oxide (NO)) and increase production of immunosuppressive IL-10 which protects cartilage from inflammation-related injury. Accelerated neotissue filling and increased synthesis of type II collagen were noticed in osteoarthritic animals that received MSC-sourced secretome. MSCderived extracellular vesicles (EVs) promoted endogenous cartilage repair and regeneration by delivering miR-320c and miR-92a-3p which restore homeostasis in bioenergetics and cell metabolism in proliferating chondrocytes.
Therapeutic potential of MSC-sourced secretome was confirmed in vivo, as well. By using an immunocompetent rat osteochondral defect model, Zhang and colleagues demonstrated that multiple intra-articular injections (one/week for 12 weeks) of human MSC-Exos promoted cartilage repair and regeneration [26]. Accelerated neotissue filling and increased synthesis of type II collagen were noticed in OA lesions. MSC-Exos-treated OA rats displayed complete restoration of cartilage and subchondral bone with characteristic features including diffuse hypercellularity, good surface regularity and less bone erosions [26]. By analyzing signaling pathways involved in chondrocyte growth and proliferation, Toh and colleagues proposed that intra-articular administration of MSC-Exos promoted endogenous cartilage repair and regeneration by restoring homeostasis in bioenergetics and cell metabolism in proliferating chondrocytes. MSC-Exos contain glycolytic enzymes phosphoglucokinase and pyruvate kinase which activity restores redox potential in chondrocytes and facilitates regeneration of OA cartilage [27]. In addition, by delivering ATPgenerating enzymes (adenylate kinase and nucleoside-diphosphate kinase), MSC-Exos may compensate reduced mitochondrial ATP production in OA chondrocytes enabling their enhanced proliferation. MSC-Exos express CD73, ecto 5-nucleotidase which degrades AMP to adenosine which, in turn, phosphorylates and activates survival kinases (Erk1/2 and Akt) [97]. Thus, when newly generated ATP was hydrolyzed to AMP, MSC-Exos, in CD73-dependent manner, converted AMP to adenosine and generated Erk1/2 and Akt-driven pro-survival signal in chondrocytes that initiated their proliferation resulting in enhanced regeneration of OA cartilage [27]. Chondrocytes cultured in the presence of MSC-derived secretome significantly reduce production of inflammatory cytokines which play detrimental role in cartilage degeneration during OA development (TNF-α, IL-1β, IL-6, nitric oxide (NO)) and increase production of immunosuppressive IL-10 which protects cartilage from inflammation-related injury. Accelerated neotissue filling and increased synthesis of type II collagen were noticed in osteoarthritic animals that received MSC-sourced secretome. MSC-derived extracellular vesicles (EVs) promoted endogenous cartilage repair and regeneration by delivering miR-320c and miR-92a-3p which restore homeostasis in bioenergetics and cell metabolism in proliferating chondrocytes.
Therapeutic potential of MSC-sourced secretome was confirmed in vivo, as well. By using an immunocompetent rat osteochondral defect model, Zhang and colleagues demonstrated that multiple intra-articular injections (one/week for 12 weeks) of human MSC-Exos promoted cartilage repair and regeneration [26]. Accelerated neotissue filling and increased synthesis of type II collagen were noticed in OA lesions. MSC-Exos-treated OA rats displayed complete restoration of cartilage and subchondral bone with characteristic features including diffuse hypercellularity, good surface regularity and less bone erosions [26]. By analyzing signaling pathways involved in chondrocyte growth and proliferation, Toh and colleagues proposed that intra-articular administration of MSC-Exos promoted endogenous cartilage repair and regeneration by restoring homeostasis in bioenergetics and cell metabolism in proliferating chondrocytes. MSC-Exos contain glycolytic enzymes phosphoglucokinase and pyruvate kinase which activity restores redox potential in chondrocytes and facilitates regeneration of OA cartilage [27]. In addition, by delivering ATP-generating enzymes (adenylate kinase and nucleoside-diphosphate kinase), MSC-Exos may compensate reduced mitochondrial ATP production in OA chondrocytes enabling their enhanced proliferation. MSC-Exos express CD73, ecto 5-nucleotidase which degrades AMP to adenosine which, in turn, phosphorylates and activates survival kinases (Erk1/2 and Akt) [97]. Thus, when newly generated ATP was hydrolyzed to AMP, MSC-Exos, in CD73-dependent manner, converted AMP to adenosine and generated Erk1/2 and Akt-driven pro-survival signal in chondrocytes that initiated their proliferation resulting in enhanced regeneration of OA cartilage [27]. Abbreviations: bone marrow (BM); adipose tissue (AT); mesenchymal stem cells (MSCs); conditioned medium (CM); exosomes (Exos); osteoarthritis (OA); interleukin (IL)-10; microRNA (miR); lncRNA KLF3 Antisense RNA 1 (KLF3-AS1).
In an analogy, Liu and colleagues recently demonstrated that therapeutic effects of MSC-Exos in cartilage regeneration were particularly related to the activity of lncRNA KLF3 Antisense RNA 1 (KLF3-AS1), which acted as a competitive endogenous RNA that segregated miRNA206 away from its target G-protein-coupled receptor kinase interacting protein 1 (GIT-1) [94,95]. GIT-1 and miRNA206 have opposite effects on chondrogenesis. While GIT-1 prevents apoptosis of chondrocytes and, therefore, promotes cartilage regeneration and chondrogenesis [100], miRNA206 inhibits proliferation of chondrocytes and enhances cartilage degradation [100]. Accordingly, upon intra-articular administration, KLF3-AS1-bearing MSC-Exos were taken up by proliferating chondrocytes in injured cartilage of OA animals and, by suppressing miRNA206-based inhibition of GIT-1 activity, prevented apoptosis and enhanced proliferative capacity of chondrocytes resulting in cartilage repair and regeneration [95]. Therefore, significantly enhanced expression of cartilage specific gene Col2A1 (which encodes the alpha-1 chain of type II collagen) and cartilage specific protein aggrecan accompanied with down-regulated expression of cartilage degrading matrix metalloproteinase (MMP)-13 resulted in increased cartilage thickness that was observed in KLF3-AS1-MSC-Exo-treated OA rats [95].

Attenuation of Inflammatory Bowel Diseases by MSC-Derived Secretome
MSCs may suppress detrimental immune response in the gut, and were, therefore, used in cell-based therapy of inflammatory bowel diseases (IBDs). However" results obtained in several clinical studies indicated that transplanted MSCs may either attenuate or aggravate colon inflammation [101][102][103]. It was concluded that engrafted MSCs polarized either in pro-inflammatory or anti-inflammatory cells in dependence of the concentration of inflammatory cytokines in the injured gut. After engraftment in the gut of patients with dominant Th1 or Th17 immune response (manifested by elevated concentration of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin (IL)-17) MSCs developed an anti-inflammatory phenotype, produced immunosuppressive IL-10 and KYN which efficiently inhibit proliferation, activation and effector function of inflammatory M1 macrophages, Th1 and Th17 cells, and alleviated CD. On contrary, after engraftment in the gut with low levels of Th1/Th17 inflammatory cytokines, MSCs adopted a pro-inflammatory phenotype, produced large amounts of inflammatory mediators which promoted migration and activation of neutrophils and effector T cells resulting in aggravation of CD [101][102][103].
In line with these findings, Yang and co-workers demonstrated that inhibition of NF-kB p65-signaling pathway in colon-infiltrated immune cells, attenuation of oxidative stress and inhibition of apoptosis were mainly responsible for beneficial effects of MSC-derived secretome in IBD therapy [29]. Significantly decreased activity of myeloperoxidase (MPO), malondialdehyde (MDA) and notably increased expression of superoxide dismutase (SOD) and glutathione (GSH) in injured colons of BM-MSC-EVs-treated animals, indicating that modulation of anti-oxidant/oxidant balance in inflamed gut had important role for BM-MSC-EVs-based therapeutic effects. Additionally, significantly reduced cleavage of caspase-3,-8 and -9, observed in injured colons of BM-MSC-EVs-treated animals, suggested that modulation of apoptosis was also, at least partially, responsible for beneficial effects of BM-MSC-sourced secretome [29].
Ubiquitin is up-regulated in colitis and its down-regulation inhibits on-going inflammation in the gastrointestinal tract [30]. In line with these findings, Wu and colleagues suggested that down-regulation of ubiquitin in inflamed gut could be responsible for MSC-Exo-based attenuation of colitis since expression of ubiquitin and ubiquitin-associated molecules (K48, K63 and FK2) were significantly decreased in DSS-treated mice after injection of UCD-MSC-Exos [107].
14 In order to avoid unwanted effects of MSC-based therapy and, at the same time, utilize their immunosuppressive potential, several research groups [28,[104][105][106] investigated therapeutic potential of MSC-sourced secretome as MSC-based, cell-free therapy for IBD (Table 4). Mao and colleagues demonstrated beneficial effects of human UCD-MSC-Exos in alleviation of dextran sodium sulphate (DSS)-induced colitis (Figure 3). MSC-Exos were detected in inflamed colons 12 hours after intravenous administration where, mainly by suppressing production of inflammatory cytokines in colon-infiltrating macrophages, attenuated on-going inflammation [28]. Among inflammatory cytokines, UCD-MSC-Exo-based therapy particularly down-regulated expression of IL-7 which promoted mucosal inflammation in the gut by acting as a mitogen and survival factor for T cells [28,105,106].
Cardiomyocytes, ECs and cardiac stem cells (CSCs) were the main cellular targets in MSC-Exos-based cardiac regeneration ( Figure 4) [109,110]. Intramyocardial injection of MSC-Exos induced cardiomyocyte proliferation and neo-angiogenesis which were manifested by significantly improved cardiac function and increased capillary density in ischemic zones of infarcted hearts [109]. MSC-Exos rescued myocardial ischaemia/reperfusion injury and reduced myocardial infarct size in experimental animals. Mechanistically, MSC-Exos increased survival of cardiomyocytes in ischemic lesions by preventing apoptosis and by inducing autophagy via AMPK/mTOR and Akt/mTOR pathways [111]. Administration of MSC-Exos resulted in up-regulation of anti-apoptotic Bcl-2, down-regulation of pro-apoptotic Bax and suppressed activity of caspase-3 in cardiomyocytes [112]. Cui and coworkers showed that MSC-Exos protected cardiomyocytes against apoptosis through the activation of Wnt/β-catenin signaling pathway since pharmacological inhibition of this cascade neutralized MSC-Exos-induced anti-apoptotic and cardioprotective effects [112]. Additionally, as recently demonstrated by Zhu and colleagues, MSC-Exos delivered miR-210 and miR-125b-5p in cardiomyocytes, and increased their survival by preventing p53 and Bak1-driven apoptosis [113,114]. Importantly, hypoxia significantly enriched miR-210 and miR-125b-5p content in MSC-Exos and enhanced their cardioprotective effects [113,114]. Significantly higher survival, smaller scar size and better cardiac function were observed in animals that received MSC-Exos obtained from MSCs which were cultured in hypoxic conditions compared to those that received Exos derived from MSCs that grew under standard culture conditions [114]. The positive effect of hypoxia on MSC-Exo-based cardioprotection was relied on expression of neutral sphingomyelinase 2 (nSMase2) which regulated miR-210 secretion [114]. Inhibition of nSMase2 activity significantly reduced miR-210 secretion and completely abrogated beneficial effects of MSC-Exos in myocardial repair [114].

17
better cardiac function were observed in animals that received MSC-Exos obtained from MSCs which were cultured in hypoxic conditions compared to those that received Exos derived from MSCs that grew under standard culture conditions [114]. The positive effect of hypoxia on MSC-Exo-based cardioprotection was relied on expression of neutral sphingomyelinase 2 (nSMase2) which regulated miR-210 secretion [114]. Inhibition of nSMase2 activity significantly reduced miR-210 secretion and completely abrogated beneficial effects of MSC-Exos in myocardial repair [114]. Modulation of angiogenesis in peri-infarcted myocardial zone was, at least partially, responsible for MSC-Exo-induced beneficial effects [115]. In line with our recently published study that emphasized the important role of stromal cell-derived factor 1 (SDF-1) in neo-angiogenesis [116], Gong and colleagues showed that SDF-1-overexpression in MSCs-Exos inhibited apoptosis of Modulation of angiogenesis in peri-infarcted myocardial zone was, at least partially, responsible for MSC-Exo-induced beneficial effects [115]. In line with our recently published study that emphasized the important role of stromal cell-derived factor 1 (SDF-1) in neo-angiogenesis [116], Gong and colleagues showed that SDF-1-overexpression in MSCs-Exos inhibited apoptosis of cardiomyocytes by promoting generation of new blood vessels in peri-infarcted myocardial zone [117]. As demonstrated by Ma and colleagues [109], miR-132 was also involved in MSC-Exo-induced neovascularization in ischemic hearts. MSC-Exo-mediated delivery of miR-132 resulted in enhanced tube formation and increased angiogenic capacity of ECs [118].
In addition to alleviation of myocardial ischemia/reperfusion injury, MSC-Exos efficiently attenuated dilated cardiomyopathy [121]. Significantly improved myocardial function, attenuated cardiac dilation and reduced apoptosis of cardiomyocytes were observed in animals that intravenously received MSC-Exos. Beneficial effects of MSC-Exos were mainly relied on their anti-inflammatory effects. MSC-Exos improved the inflammatory microenvironment in the hearts by regulating function of macrophages, which were crucially important for the development of myocardial inflammation in dilated cardiomyopathy. Systemic injection of MSC-Exos remarkably attenuated the total number of pro-inflammatory macrophages in the hearts and significantly decreased serum concentration of macrophage-derived inflammatory cytokines and chemokines which reduced influx of circulating inflammatory cells in the MSC-Exos treated hearts [121].
Beneficial effects of MSC-Exos in glaucoma treatment were relied on activity of MSC-derived miRNAs [43]. Knockdown of Argonaute2 protein, which is crucially important for miRNA function, significantly attenuated BM-MSC-Exo-induced effects [124]. RNA sequencing revealed that more than 40 miRNAs were up-regulated in BM-MSC-Exos, compared to fibroblast-derived Exos, and, among them, miR-17-92, miR-21 and miR146a were designated as the most important for regeneration of RGCs in glaucomatous eyes [122,123]. Expression of phosphatase and tensin homolog (PTEN), which is an important suppressor of RGC axonal growth and survival, were regulated by miR-17-92 and miR-21 while miR-146a modulated expression of EGFR involved in inhibition of axon regeneration [43]. 19 therapeutic effects of BM-MSC-Exos in glaucoma treatment were similar to those observed in BM-MSC-treated animals [43,[122][123][124]. Importantly, these beneficial effects were not noticed after intravitreal injection of fibroblasts-derived Exos, indicating specific therapeutic potential of MSCs-Exos in RGCs regeneration and glaucoma treatment [124]. However, beneficial effects of BM-MSC-Exos were only observed when BM-MSC-Exos were continuously injected (at least once per week) in glaucomatous eyes while longer delays between treatments completely abrogated MSC-Exodependent effects [124]. Beneficial effects of MSC-Exos in glaucoma treatment were relied on activity of MSC-derived miRNAs [43]. Knockdown of Argonaute2 protein, which is crucially important for miRNA function, significantly attenuated BM-MSC-Exo-induced effects [124]. RNA sequencing revealed that more than 40 miRNAs were up-regulated in BM-MSC-Exos, compared to fibroblast-derived Exos, and, among them, miR-17-92, miR-21 and miR146a were designated as the most important for By using animal model of laser-induced retinal injury, Yu and co-workers demonstrated that MSC-Exos supply injured retinas with immunomodulatory factors which results in alleviation of retinal inflammation [34]. Attenuated laser-induced retinal injury, observed in MSC-Exo-treated eyes, was accompanied by an increased number of photoreceptor cells and significantly reduced number of inflammatory cells, particularly CD68+ macrophages. Cellular make-up of the retinas revealed that MSC-Exos suppressed MCP-1-dependent migration of monocytes in injured retinas and attenuated TNF-α-driven retinal inflammation. Expression of macrophage-derived TNF-α and MCP-1 were down-regulated in MSC-Exo-treated retinas. Application of MCP-1 completely diminished immunosuppressive and therapeutic effects of MSC-Exos and significantly aggravated macrophage-driven inflammation and laser induced injury [34].
MSC-Exos suppressed detrimental immune response in the eye during the attenuation of experimental autoimmune uveitis (EAU) and corneal injury [122]. As demonstrated by Bai and colleagues, periocular injection of MSC-Exos attenuated EAU by reducing MCP-1 and CCL21-dependent influx of neutrophils, NK cells, macrophages and T cells in inflamed retinas [35]. Among effector T cells, MSC-Exos selectively prevented influx of CXCR3-expressing, IFN-γ producing Th1 and CCR5-expressing IL-17 producing Th17 cells in inflamed retinas, without affecting migration of immunosuppressive T regs [35]. Similar to these results were findings obtained by Shigemoto-Kuroda and colleagues [125] who demonstrated that suppression of Th1 and Th17 immune response was a consequence of MSC-Exo-based attenuation of antigen-presenting function of DCs. Flow cytometry analysis of MSC-Exo-treated DCs revealed down-regulated expression of co-stimulatory molecules (CD40, CD80 and CD86) and reduced expression of MHC class II molecules [125]. Additionally, the transcript levels of Th1 (IL-12, IFN-γ) and Th17-related inflammatory cytokines (IL-1β, IL-6, and IL-17A) were significantly lower in the eyes of MSCs-Exos-treated mice when compared to the vehicle-treated controls, indicating that the main mechanism of MSC-Exos-mediated attenuation of EAU was relied on suppression of DC-driven generation of Th1 and Th17 immune response [35,125].
In a similar manner as in EAU, detrimental immune response has crucially important role in the pathogenesis of corneal injury and DED, multifactorial diseases of the ocular surface and tears that result in visual disturbances [122]. It is well known that IL-1β-producing macrophages orchestrate influx of circulating leukocytes in injured corneas while Th17 cell-derived IL-17A and IL-22 regulate progression of DED [126][127][128]. Accordingly, suppression of IL-1β-driven inflammation in corneal tissue and attenuation of Th17 immune response resulted in alleviation of corneal injury and DED [122]. MSC-derived IL-1Ra attenuate production of inflammatory cytokines (IL-1β and TNF-α) in M1 macrophages and promotes their polarization towards immunosuppressive, IL-10-producing M2 phenotype [129]. Similarly, DCs cultured in the presence of MSCs-derived IDO-1 and growth-related oncogene (GRO) developed tolerogenic and immunosuppressive phenotype and, instead of Th17-related inflammatory cytokines, produced large amounts of anti-inflammatory IL-10 [60,130]. In line with these observations, we recently developed an immunomodulatory ophthalmic solution ("Exosomes Derived Multiple Allogeneic Proteins Paracrine Signaling (Exosomes D-MAPPS)") whose activity is based on the capacity of MSC-Exos to suppress immune response in IL-1Ra, GRO and IDO-1/KYN-dependent manner having beneficial effects in the treatment of corneal injuries an DED [43,131].
In addition to degenerative and inflammatory diseases, MSC-derived secretome efficiently alleviated Mucopolysaccharidosis VII (Sly Syndrome), corneal congenital metabolic disease caused by a mutation of β-glucuronidase, enzyme required for the degradation of glycosaminoglycans (GAGs) [132].
Coulson-Thomas and colleagues demonstrated that, after intraocular administration, UCD-MSC-Exos delivered β-glucuronidase into the keratocytes and enabled degradation of accumulated GAGs. These findings indicate that UCD-MSC-Exos should be further explored as new, cell-free vehicles for enzyme substitution therapy of inherited metabolic diseases.

Type of Secretome
Similarly, Dong and co-workers recently demonstrated that systemic injection of miR-133b-bearing MSC-Exos promoted recovery from spinal cord injury (SCI) by promoting regeneration of axons through the activation of survival Erk1/2 and Stat-3 signaling pathways in neurons. Importantly, miR-133b-bearing-MSC-Exos significantly improved recovery of hindlimb locomotor function in experimental rats [134], indicating that these MSC-EVs should be further explored as new, cell-free therapeutic agents for the treatment of SCI ( Figure 6). In addition to their direct neuroprotective effects of injured neurons, MSC-Exos are also able to modulate microenvironment of spinal cord lesions through their anti-inflammatory and proangiogenic effects [135,136]. As evidenced by Huang and colleagues, systemic application of MSC-Exos promoted functional recovery following SCI by inducing neo-angiogenesis and through the suppression of TNF-α and IL-1β-driven inflammation [135]. Furthermore, MSC-Exo treatment enhanced production of immunosuppressive IL-10 which suppressed neurotoxic A1 astrocytes [135]. In line with these findings, Wang and co-workers showed that MSC-Exos may prevent proinflammatory properties of A1 astrocytes by inhibiting nuclear translocation of p65 subunit of NF-κB, which is crucially important for the generation of inflammatory phenotype in these cells [137]. In addition to N1 astrocytes, macrophages were the main cellular targets in MSC-Exo-based immunomodulation of SCI [136,138]. After intravenous administration, MSC-Exos accumulated at the site of SCI where promoted polarization of inflammatory M1 macrophages into immunosuppressive M2 phenotype. Accordingly, enhanced presence of CD206-expressing and IL-10-producing M2 macrophages and reduced number of TNF-α and IL-1β-producing M1 macrophages were observed in spinal cord lesions of MSC-Exo-treated animals [136,138]. Having in mind that TNF-α and IL-1β-driven inflammation results in severe neuropathic pain, Shiue and colleagues investigated therapeutic potential of MSC-Exos in attenuation of nerve-injury induced pain [139]. They demonstrated that continuous intrathecal infusion of human UCD-MSC-Exos Similarly, systemic injection of miR-133b-bearing MSC-Exos promoted recovery from SCI by promoting regeneration of axons through the activation of survival Erk1/2 and Stat-3 signaling pathways in regenerating neurons. After intravenous administration, MSC-Exos accumulated at the site of SCI and promoted generation of immunosuppressive M2 macrophages which, in IL-10-dependent manner, suppressed activation of neurotoxic A1 astrocytes through the inhibition of NF-kB. In similar manner, via down-regulation of NF-κB p65 signaling, MSC-EVs reduced migratory capacities of pericytes and maintained structural integrity of blood-spinal cord barrier (BSCB).
In addition to their direct neuroprotective effects of injured neurons, MSC-Exos are also able to modulate microenvironment of spinal cord lesions through their anti-inflammatory and pro-angiogenic effects [135,136]. As evidenced by Huang and colleagues, systemic application of MSC-Exos promoted functional recovery following SCI by inducing neo-angiogenesis and through the suppression of TNF-α and IL-1β-driven inflammation [135]. Furthermore, MSC-Exo treatment enhanced production of immunosuppressive IL-10 which suppressed neurotoxic A1 astrocytes [135]. In line with these findings, Wang and co-workers showed that MSC-Exos may prevent pro-inflammatory properties of A1 astrocytes by inhibiting nuclear translocation of p65 subunit of NF-κB, which is crucially important for the generation of inflammatory phenotype in these cells [137]. In addition to N1 astrocytes, macrophages were the main cellular targets in MSC-Exo-based immunomodulation of SCI [136,138]. After intravenous administration, MSC-Exos accumulated at the site of SCI where promoted polarization of inflammatory M1 macrophages into immunosuppressive M2 phenotype. Accordingly, enhanced presence of CD206-expressing and IL-10-producing M2 macrophages and reduced number of TNF-α and IL-1β-producing M1 macrophages were observed in spinal cord lesions of MSC-Exo-treated animals [136,138]. Having in mind that TNF-α and IL-1β-driven inflammation results in severe neuropathic pain, Shiue and colleagues investigated therapeutic potential of MSC-Exos in attenuation of nerve-injury induced pain [139]. They demonstrated that continuous intrathecal infusion of human UCD-MSC-Exos achieved excellent preventive and reversal effects for nerve ligation-induced pain. Analgesic effects of MSC-Exos were relied on the delivery of neurotrophins (BDNF, glial cell line-derived neurotrophic factor (GDNF)) and immunosuppressive factors (IL-10) in the neurons and glial cells [139].
As recently revealed by Lu and colleagues, systemic administration of BM-MSC-EVs improved motor function in SCI-treated animals by preventing disruption of the blood-spinal cord barrier (BSCB) [140]. Since pericytes play a pivotal role in maintaining the structural integrity of BSCB, BM-MSC-EVs increased total number of pericytes in BSCB by reducing their migratory capacities via down-regulation of NF-κB p65 signaling [140].

Clinical Studies Addressing Therapeutic Potential of MSC-Derived Secretome
Although results obtained in animal models suggested beneficial effects of MSC-sourced secretome, only several clinical studies confirmed regenerative and immunomodulatory potential of MSC-CM and MSC-derived EVs. Administration of MSC-derived secretome efficiently improved clinical outcomes in patients suffering from severe alveolar bone atrophy, alopecia and graft-versus-host disease (GvHD) [141][142][143][144]. Importantly, adverse effects have not been reported in patients that received MSC-sourced secretome, indicating that local and systemic injection of MSC-CM and MSC-Exos is safe therapeutic approach [141][142][143][144].
In the case of alveolar bone regeneration, eight patients received either porous pure beta-tricalcium phosphate or shell-shaped atelocollagen sponge scaffold grafts soaked in the BM-MSC-CM [141]. Radiographic and histological evaluation revealed mineralization, early bone formation and reduced infiltration of inflammatory cells in patients that received BM-MSC-CM-containing scaffold grafts. Among MSC-derived immunomodulatory and trophic factors, VEGF, TGF-β, and HGF contributed to the beneficial effects of BM-MSC-secretome in bone regeneration [141].
Results obtained in clinical trials addressing alopecia [142] and Female Pattern Hair Loss (FPHL) [143] revealed that AT-MSC-CM may represent a new therapy for hair regeneration. Significantly increased hair density was observed in 22 patients with alopecia (11 men and 11 women) and in 27 patients with FPHL that intradermal received AT-MSC-CM (0.02 mL/cm 2 ). Approximately a total volume of 3 to 4 mL of AT-MSC-CM was administered during each session of treatment. Patients received intradermal treatment of AT-MSC-CM every 3 to 5 weeks for a total of 6 sessions. Among MSC-derived growth factors, elevated levels of HGF, FGF-1, IL-6, VEGF and TGF-β were measured in AT-MSC-CM. Importantly, AT-MSC-CM was well tolerated and no side effects were observed in 49 patients that received multiple intradermal injections of AT-MSC-derived secretome [142,143].
MSC-Exos significantly improved symptoms of GvHD in a patient who suffered from treatment-refractory GvHD [144]. According to the application regime of MSCs in GvHD patients (0.4-9.0 × 10 6 MSCs/kg body weight) [145] the amount of MSC-Exos obtained from the supernatant of 4 × 10 7 MSCs was used as a 1 therapeutic unit [144]. To reduce the risk of potential side effects, only a tenth of an MSC-Exo unit was initially administered. Since no side effects were observed, unit amounts were gradually increased and 4 therapeutic units were administered every 2-3 days in next several months. Systemic injection of MSC-Exos was well tolerated and no side effects were observed during 7-month follow-up period. Among MSC-derived immunosuppressive factors, IL-10 and TGF-β were noticed in the highest concentrations in MSC-Exos. Accordingly, MSC-Exos impaired capability of patient's PBMNCs to produce inflammatory cytokines (IL-1β, IFN-γ and TNF-α) and attenuated on-going inflammation in gut and skin [144]. Reduction of diarrhea volume and attenuation of cutaneous and mucosal symptoms associated with GvHD were observed two weeks after initial administration of MSC-Exos and were stable in next 4 months, indicating a long-lasting therapeutic effect of MSC-Exos [144].
In line with these findings, researchers from the Isfahan University of Medical Sciences decided to elucidate safety and efficacy of MSC-Exos on disability of patients with acute ischemic stroke.
This clinical trial has not been started yet. Patients will, one month after ischemic injury, receive allogenic miR-124-expressing MSC-Exos via stereotaxis. Incidence of treatment-emergent side effects (stroke recurrences, brain edema, seizures) and improvement of disability will be monitored during this study (NCT03384433).
Similarly, researchers from the Punta Pacifica Hospital of Panama City decided to elucidate safety and efficacy of allogeneic UCD-MSC-derived trophic factors (MTF) in adult asthmatic patients. This study is still recruiting patients who will intra-nasally receive MTF once per week for a period of 4 weeks. Side effects as well as alterations of lung function will be monitored during one month follow up (NCT02192736).

Conclusions and Future Perspectives
Results obtained in experimental and clinical studies suggest that MSC-derived secretome represents a promising therapeutic tool for the treatment of degenerative and inflammatory diseases. Importantly, administration of MSC-CM and MSC-EVs were as effective as transplantation of the corresponding MSCs in attenuation of acute and chronic inflammatory diseases of gastrointestinal, respiratory, cardiovascular and central nervous system [19][20][21][22]28,29,[31][32][33]37,72,74,[97][98][99][122][123][124]127,137]. Beneficial effects of MSC-sourced secretomes rely on their capacity to deliver genetic material, growth and immunomodulatory factors to the target cells enabling activation of anti-apoptotic and pro-survival pathways which results in enhanced tissue repair and regeneration (Figure 7). recurrences, brain edema, seizures) and improvement of disability will be monitored during this study (NCT03384433). Similarly, researchers from the Punta Pacifica Hospital of Panama City decided to elucidate safety and efficacy of allogeneic UCD-MSC-derived trophic factors (MTF) in adult asthmatic patients. This study is still recruiting patients who will intra-nasally receive MTF once per week for a period of 4 weeks. Side effects as well as alterations of lung function will be monitored during one month follow up (NCT02192736).

Conclusions and Future Perspectives
Results obtained in experimental and clinical studies suggest that MSC-derived secretome represents a promising therapeutic tool for the treatment of degenerative and inflammatory diseases. Importantly, administration of MSC-CM and MSC-EVs were as effective as transplantation of the corresponding MSCs in attenuation of acute and chronic inflammatory diseases of gastrointestinal, respiratory, cardiovascular and central nervous system [19][20][21][22]28,29,[31][32][33]37,72,74,[97][98][99][122][123][124]127,137]. Beneficial effects of MSC-sourced secretomes rely on their capacity to deliver genetic material, growth and immunomodulatory factors to the target cells enabling activation of antiapoptotic and pro-survival pathways which results in enhanced tissue repair and regeneration ( Figure 7). Results obtained in experimental studies suggest that MSC-derived secretome represents a promising therapeutic tool for the treatment of degenerative and inflammatory diseases. Beneficial effects of MSC-sourced secretomes rely on their capacity to deliver neurotrophins (brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), hepatocyte growth factor (HGF), miR-17-92, miR-21, miR-124, miR-133b, miR146a which enable regeneration of injured liver, brain, spinal cord and eye. MSC-derived secretomes contain immunomodulatory factors which inhibit proliferation and activation of inflammatory immune cells and promote expansion of Figure 7. Molecular mechanisms responsible for beneficial effects of MSC-derived secretome in tissue repair and regeneration. Results obtained in experimental studies suggest that MSC-derived secretome represents a promising therapeutic tool for the treatment of degenerative and inflammatory diseases. Beneficial effects of MSC-sourced secretomes rely on their capacity to deliver neurotrophins (brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), hepatocyte growth factor (HGF), miR-17-92, miR-21, miR-124, miR-133b, miR146a which enable regeneration of injured liver, brain, spinal cord and eye. MSC-derived secretomes contain immunomodulatory factors which inhibit proliferation and activation of inflammatory immune cells and promote expansion of immunosuppressive cells resulting in alleviation of inflammation-related tissue injury. MSC-sourced secretomes are enriched with angiomodulatory factors (stromal cell derived factor-1 (SDF-1), miR-132, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF)) that promote angiogenesis and neo-vascularization in ischemic regions of brain and heart enhancing survival of injured neurons and cardiomyocytes. However, it should be noted that there are still several issues which limit potential clinical use of MSC-derived secretome. Therapeutic potential of MSC-sourced secretome depends on functional properties of MSCs from which it was obtained. Although MSCs have low expression of MHC molecules, several lines of evidence indicated that transplantation of allogeneic MSC can induce measurable allogeneic immune responses in MHC-mismatched recipients [146][147][148][149]. However, immunogenicity of MSC-sourced secretome is still a matter of debate. Lou and colleagues proposed that MSC-Exos are less immunogenic than their parent MSCs because of lower content in membrane-bound proteins including tetraspanins (CD81, CD63 and CD9), heat-shock proteins (HSP60, HSP70 and HSP90), programmed cell death 6-interacting protein and tumor susceptibility gene 101 [13]. In line with these observations are findings recently reported by Kordelas and coworkers who demonstrated that multiple injections of BM-MSC-Exos, obtained from four unrelated donors, did not evoke allogeneic immune response in MHC-mismatched recipient [144]. Nevertheless, evidence recently provided by Liu and coworkers indicated that potentially immunogenic proteins such as MHC molecules can also be transferred via EVs [150]. This raises the important safety concern for administration of MSC-EVs in MHC-mismatched recipients since MHC-bearing MSC-EVs could provoke detrimental allogeneic immune response. It should be emphasized that there is still no clear evidence that MSC-EVs are able to transfer MHC molecules to target cells that could result in generation of allogeneic immune response. Therefore, future experimental studies should be designed to investigate the influence of MHC-bearing MSC-EVs on immune response of MHC-mismatched recipients in order to delineate immunogenicity of MSC-derived secretome.
Additionally, since sub-populations of MSCs differ in their capacity for differentiation and immunomodulation, heterogeneity of MSC-derived secretomes may cause diverse effects on their target cells. MSCs should be exposed to culture conditions which reflect a specific inflammatory microenvironment of the tissue that is going to be regenerated by MSC-derived secretome. Methods used to precondition MSCs in stimulating their functional properties, such as hypoxia and cytokine priming, significantly modify content and therapeutic effects of MSC-sourced secretome. Therefore, future experimental and clinical studies should precisely define protocols for generation of MSC-derived secretome for each of MSC subpopulations and for particular pathological conditions before MSC-sourced secretome will be offered worldwide as a universal human remedy.

Conflicts of Interest:
The authors declare no conflict of interest.